Constitutive producer of a thermostable glucose isomerase

The maintenance medium (SCM) for all microorganisms herein consisted of 1% yeast autolysate, 1% casein enzymatic hydrolysate, 0.5% beef extract, 1 millimolar magnesium sulfate, and 0.05 molar potassium phosphate with the pH adjusted to 7.10 with sodium hydroxide before autoclaving. Bacteria were transferred regularly onto a 2% agar medium containing the above ingredients as an inoculum for liguid cultures. The strains were generally incubated for 48 hours in a liquid culture at 45° C. for thermophilic organisms, 30° C. otherwise, with agitation (200 rpm) in a New Brunswick Controlled Environment Shaker.

After 48 hours, the 50 ml cultures (contained in 250 ml baffled Erlenmeyer flasks) were harvested and washed free of spent culture broth by centrifugation (4° C. at 15,000 rpm for 10 minutes). A portion of the cell pellet was dried by microwave and % solids calculated, using a CEM Corp. Model AVC-MP instrument. The remaining (wet) cells were resuspended in 30 ml of 0.02 M potassium phosphate buffer, pH 7.0, containing 0.85% sodium chloride. This material was sonicated for 1 minute at low power with a Labline sonifier.

For the glucose isomerase assay procedure, 0.2 ml of the sonicated cell slurry was diluted to 1 ml with distilled H 2 O. This 1 ml sample was added to 3 mls of the substrate solution which consists of 45% fractose (W/V), 0.05 M imidazole buffer at pH 7.5, 5×10 -3 M MgSo 4 , and 5×10 -4 M CoCl 2 . After thorough mixing, the reaction mixture was incubated at 60°C. for a 60 minute duration. The reaction was terminated by quenching with the addition of 1 ml of 0.1 N HCl with mixing, and chilling on ice. The enzyme activity was determined by measurement of glucose concentration using a Beckman Glucose Analyzer. One unit of GI activity is equivalent to the formation of 1 umole of glucose per ml per minute.

Cultures of Streptomyces thermovulgaris, ATCC 19284, and Streptomyces thermoviolaceus, ATCC 19283, were examined for glucose isomerase production with the results summarized in the following table.

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Glucose Isomerase Activity of Thermophilic Streptomyces

GI activity, units/g dry weight cells

Strain No xylose 0.5% xylose added

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S. thermovulgaris,

ca. 10 190

ATCC 19284

S. thermoviolaceus,

ca. 10 60

ATCC 19283

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As the table shows, both thermophilic organisms produce glucose isomerase at relatively low levels even with xylose in the growth medium.

Having selected two thermophilic microorganisms which are GI producers, it was necessary to determine the thermal stability of their glucose isomerase, especially relative to other isomerases. For the purpose of this application, a thermostable GI is one which loses less than 10% of its isomerase activity when heated for 5 minutes at 90° C. in a solution of 42% fructose at pH 7.0 relative to its activity after heating for 5 minutes at 80° C. under otherwise identical conditions.

Crude extracts of glucose isomerase were prepared by sonication of harvested wet cells with subsequent dialysis into 0.1 molar potassium phosphate, pH 7.0, containing 1 millimolar magnesium sulfate. Table 2 summarizes the results when thermal stability was measured in a phosphate buffer at pH 7.0. In all cases the enzyme solution was heated at the indicated temperature for 5 minutes.

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Thermal Stability of Various GI's in Phosphate Buffer

GI activity (% of original)

after 5 minutes heating at

Strain 80° C.

90° C.

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S. alboduncus (ATCC 14698)

84 4

S. albovinaceus (ATCC 15823)

72 10

S. omiyaensis (ATCC 27454)

53 4

S. thermoviolaceus (ATCC 19283)

98 60

Actinoplanes missouriensis

103 23

(NRRL B 3342)

S. thermovulgaris (ATCC 25501)

78 6

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As the table clearly shows the isomerase from S. thermoviolaceus is by far the most stable to heat denaturation of those tested. Since it was previously known that thermostability in fructose solutions does not always parallel that observed in a dilute phosphate buffer, thermostability was determined by heating crude GI extracts in a solution of 42% fructose at pH 7.0. Results are summarized in the following Table 3.

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Thermal Stability of Various GI's

in High Fructose Corn Syrup

GI activity (% of original)

after 5 minutes heating at

Strain 80° C.

90° C.

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S. alboduncus (ATCC 14698)

82 59

S. albovinaceus (ATCC 15823)

92 71

S. omiyaensis (ATCC 27454)

120 84

S. thermoviolaceus (ATCC 19283)

69 69

A. missouriensis (NRRL B 3342)

90 76

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These data clearly show little, if any, additional loss of activity resulting from heating GI from S. thermoviolaceus at 90° C. as compared to 80° C., thereby demonstrating the superior thermal stability of the GI from this species relative to the other enzymes examined.

Having demonstrated the superior thermal stability of the GI from S. thermoviolaceus, it was now desired to obtain a mutant strain which overproduced glucose isomerase constitutively.

Mutagenesis was begun using the parent strain Streptomyces thermoviolaceus, ATCC 19283, in a liquid culture broth for approximately 24 hours. Ten ml of the culture was washed free of broth under sterile conditions and the wet cell pellet was resuspended in 10 ml of 0.2 M tris(hydroxymethylamino)methane hydrochloride at a pH of 7.5. Ethyl methanesulfonate was added to the cell suspension in a concentration of 0.15 ml per 10 ml suspension and the cells were then incubated, with agitation, at 30° C. for 2.5 hours. After this time the EMS treated cells were added to 50 ml of the maintenance medium and permitted to grow for 48 hours.

Screening of the mutagenized cells involved transferring the organisms from liquid cultures to agar plates so that individual colonies could be evaluated for enzyme production. To achieve this, 2.5 ml aliquots of the liquid culture were briefly (10 seconds) sonicated at low power. This procedure breaks up the mycelial strands and releases individual mutated cells. These aliquots were then recombined and diluted to a concentration of 40% glycerol and stored at -70° C. until used.

Dilutions of these EMS treated cells were then plated onto an agar medium to give approximately 200 colonies per plate. These colonies were assayed for constitutive production of glucose isomerase. The mutant microorganism Streptomyces thermoviolaceus NRRL 15615 was founded by this procedure. Comparison of GI production by the parent and mutant strains are shown in the accompanying table.

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Comparison of GI Production

GI, units/g

Microorganism 1.0% Xylose

dry cells

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ATCC 19283, parent

present 60

absent 10

NRRL 15615, mutant

present 1500

absent 1500

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Medium optimization for glucose isomerase production by the mutant strain NRRL 15615 was performed by adding xylose, corn steep liquor, or yeast extract to a base medium which contained 50 mM phosphate buffer, pH 7.0, 3 mM MgSO 4 , 2 g/L NaNO 3 , 1 mM CaCl 2 , and 2 g/L (NH 4 ) 2 SO 4 . Results are shown in the following table.

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Medium Optimization for GI Production

by S. Thermoviolaceus, NRRL 15615

Addend (1%) Presence/Absence

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Xylose + - + -

Corn steep liquor

+ + - +

Yeast extract + + + -

GI activity, 1500 1500 1000 1100

units/g dry weight

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Morphological characteristics of the parent strain and its mutant in various media after 3 days at 42° C. are summarized in Table 6.

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Morphology of S. Thermoviolaceus

Medium

(agar-based, NRRL

20 g/l) ATCC 19283 15615

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1. SCM 1-2 cm flat colonies, deep violet;

same

aromatic, almost pleasing odor;

white coating covering most colonies

2. LB.sup.a 0.5 cm flat colonies; no color or

same

odor

3. malt Isolated single colonies were white,

same

extract.sup.b

crowded colonies were yellow, dense

inoculum smear was violet; aromatic

odor

4. M9 + 1 cm colonies, volcanic shape, sur-

similar, but

casamino rounded by black-gray color

color more

acids.sup.c whitish-

gray

5. M9 + no significant growth over M9 salts

same

dextran.sup.d

alone (i.e., no dextran supplementa-

tion); very faint, flat, 0.5 cm

colonies

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.sup.a Contains per liter 10 g tryptone, 5 g yeast extract, and 10 g NaCl

.sup.b Bacto malt extract agar prepared per manufacturer's (Difco)

instructions.

.sup.c Contains per liter 6 g Na.sub.2 HPO.sub.4, 3 g KH.sub.2 PO.sub.4,

0.5 g NaCl, 1 g NH.sub.4 Cl; plus MgCl.sub.2 (1 mM), CaCl.sub.2 (0.1 mM),

and 2 g casamino acids.

.sup.d Similar to that above, with 1% dextran replacing the casamino

acids.

Table 6 shows the mutant and parent strains manifest essentially similar growth morphology on five different media. In lieu of testable genetic markers, this is the most convincing evidence that the mutant strain is indeed derived from the parent strain and is not a fortuitous contaminant.

The glucose isomerase from the mutant microorganism of this invention may be used to isomerize glucose to fructose. This may be done either homogeneously, where a solution of GI and glucose are permitted to remain in contact for a sufficient time to achieve the desired conversion, or heterogeneously, where the enzyme is supported on a suitable matrix and a solution of glucose is contacted with the immobilized enzyme, which is generally as a fixed bed. Contact temperatures from about 40° C. to about 90° C. are common, with a temperature between about 50° and 80° C. more desirable for the thermostable GI of this invention. Such isomerization is conducted at a pH between about 6.5 and 9.5, and more usually between about 7.0 and 8.5. The fructose is then recovered by suitable means, as by chromatographic absorption.